The long-term goal of this project is to define the molecular mechanisms that underlie actin assembly and understand how they contribute to cell motility. Our approach is to identify components, understand how they interact with each other in vitro using purified proteins or cell extracts, define mutations that selectively affect one interaction, and then test the biological activity of that mutant in a cell system from which the endogenous component has been depleted. The Aims all represent the continuation of ongoing projects in the lab. Aim 1 is to understand how cortactin contributes to actin assembly and cell motility. Cortactin is a prominent substrate for Src, the significance of which is not understood. We discovered that cortactin has a specific essential role in the osteoclast, which requires an actin-based sealing zone to resorb bone. With this system, we will examine the roles of Src phosphorylation and other biochemical functions of cortactin. Aim 2 is to understand how capping protein, the major capper of barbed ends of actin filaments in eukaryotes, binds to actin. We will investigate the molecular basis of the interaction and use that information to understand the physiological significance of the interaction in cells. We will test a new hypothesis, the wobble model, which changes our view of the dynamic nature of a capped barbed end. Aim 3 is to investigate the molecular basis and physiological significance of the interaction of capping protein with a number of proteins hypothesized to regulate and target capping protein in cells. The proteins include CARMIL,V1/myotrophin, twinfilin, casein kinase 2, CD2AP / Cin85, and nebulin. Relevance to public health: This research will help us understand how cells control their shape and move around the body, which are important features of development, cancer, and the response to infection. This fundamental information may be the basis for the development of new therapies for human diseases involving the molecules and processes that are discovered and illuminated.